Abstract
The adenovirus (Ad) early transcription unit E3 encodes immunosubversive functions. The E3 transmembrane proteins 10.4 and 14.5 form a complex that down-regulates the epidermal growth factor receptor and apoptosis receptors from the cell surface by diverting them to endosomes/lysosomes for degradation. The latter process protects infected cells from ligand-induced apoptosis. The mechanism by which 10.4-14.5 mediate re-routing remains elusive. We examined the role of putative YXX Phi and dileucine (LL) transport motifs within Ad2 10.4-14.5 for target protein modulation. By generating stable E3 transfectants expressing 10.4-14.5 proteins with alanine substitutions in these motifs, we show that 3 of the 5 motifs are essential for functional activity. Whereas tyrosine 74 in 14.5 appears to be important for efficient 10.4-14.5 interaction, the 122YXX Phi motif in 14.5 and the dileucine motif Leu 87-Leu88 in 10.4 constitute genuine transport motifs: disruption of either motif abolished binding to the cellular adaptor proteins AP-1 and AP-2, as shown by surface plasmon resonance spectroscopy, and caused missorting, dramatically altering cell surface appearance and the intracellular location of viral proteins. Fluorescence-activated cell sorter analysis and immunofluorescence data provide evidence that Tyr122 in 14.5 is essential for rapid endocytosis of the 10.4-14.5 complex, whereas the 10.4LL motif acts down-stream and protects 10.4-14.5 from extensive degradation by rerouting it into a recycling pathway. Infection of primary cells with adenoviruses carrying the relevant point mutations confirmed the crucial role of these transport motifs for down-regulation of Fas, TRAIL-R1, TRAIL-R2, and epidermal growth factor receptor. Thus, two distinct transport motifs present in two proteins synergize for efficient target removal and immune evasion.
Highlights
Proteins encoded within the early transcription unit E3 of human adenoviruses (Ads)1 play a key role in immune evasion [1,2,3,4]
10.4 –14.5 were reported to contribute to inhibition of tumor necrosis factor-induced cytolysis. This is not achieved by tumor necrosis factor receptor modulation but rather by inhibition of tumor necrosis factor-induced translocation of cytosolic phospholipase A2 and possibly nuclear factor B [3, 11, 17, 18]. 10.4 –14.5 removes the epidermal growth factor receptor (EGFR) from the cell surface by diverting constitutively internalized receptors into a degradation compartment (19 – 21)
From EE, the EGFR may rapidly return to the cell surface, whereas a significant pool of EGF-EGFR complexes is sorted to the inner vesicles of a late endosomal compartment, designated multivesicular bodies (MVBs)
Summary
Adenoviruses; AP, adaptor proteins; EE, early endosomes; ER, endoplasmic reticulum; EGFR, epidermal growth factor receptor; FACS, fluorescence activated cell sorting; Lamp, lysosome-associated membrane protein; LE, late endosomes; MVBs, multivesicular bodies; RID, receptor internalization and degradation; TGN, trans-Golgi network; TRAIL, tumor necrosis factor-related apoptosis inducing ligand; wt, wild-type; IP, immunoprecipitation; LL, dileucine; mAb, monoclonal antibody; SPR, surface plasmon resonance; Baf, bafilomycin A1; aa, amino acid(s). Both viral proteins contain sequence elements in their cytoplasmic tails that conform to consensus transport motifs (Fig. 1): three putative YXX⌽ motifs (where Y is a tyrosine, X any amino acid (aa), and ⌽ (an aa with a bulky hydrophobic side chain) are present in 14.5 (Fig. 1B), whereas 10.4 proteins generally display two potential dileucine (LL) motifs (Fig. 1C) Both tyrosine- and dileucine-based motifs have been shown to trigger rapid internalization from the cell surface, specify sorting to lysosomes or specialized endosomal/lysosomal compartments; they mediate trafficking to the trans-Golgi network (TGN) as well as sorting to the basolateral plasma membrane of polarized epithelial cells [35,36,37]. Our findings illustrate the highly sophisticated cooperation between two viral proteins, each providing a distinct transport signal required for efficient target removal and immune evasion
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